Cell motility, certain membrane trafficking events, and cell division all rely on regulated actin assembly. The proposed studies will reveal underlying principles governing cortical actin cytoskeleton assembly dynamics. A large collection of site-directed mutants in actin, Arp2, Arp3, cofilin, profilin and Cdc42, and null alleles of genes encoding nonessential actin-binding proteins such as Aip1, coronin and twinfilin, will be analyzed using two powerful in vivo fluorescence assays of actin assembly dynamics. For one assay, effects of the mutants on an actin-dependent endocytosis pathway consisting of a series of discrete steps will be determined. For the second assay, in vivo "treadmilling" rates will be measured using Fluorescence Recovery After Photobleaching (FRAP). Hypotheses generated from these studies will be tested using biochemical treadmilling and Arp2/3-mediated actin assembly assays. The Arp2/3 complex, with particular attention on the nucleotide and novel mechanisms of activation and regulation, will be studied in detail. A central unsolved question concerns roles for the ATPs associated with Arp2 and Arp3. Based on studies of actin, nucleotide states could influence such vital aspects of Arp2/3 complex function as its activation, branching and de-branching activities, and interactions with regulatory factors. Because Arp2 and Arp3 are closely related to actin, and because actin nucleotide-binding pocket mutants have provided valuable insights into regulation of cytoskeletal dynamics, a similar set of mutants was constructed in Arp2 and Arp3. Effects of these mutants will be tested in vivo and in a battery of in vitro tests for nucleation activity, interactions with different activators, and branching and de-branching reactions. In addition, the functional properties of four Arp2/3 activators, with a particular emphasis on the type I myosins, will be investigated. Numerous proteins that interact with each of these four activators have been identified, and the effects of these proteins, post-translational modifications, and lipids on their activating activities will be tested. The proteins under investigation are central to pathogen invasion and to metastasis of cancer cells. Aberrations in actin organization are hallmarks of cancer. Rational approaches to treatment and prevention of these disease states therefore depend on understanding principles of actin assembly regulation.